Optimising the collection of female genital tract fluid for cytokine analysis in pregnant women

Introduction: To better understand the immunology of pregnancy, study of female genital tract fluid (FGF) is desirable. However the optimum method of collection of FGF in pregnant women for immunological methods, specifically cytokine measurement, is unknown. Methods: A prospective study of HIV-uninfected pregnant women comparing two methods of FGF collection: polyvinyl acetal sponge collection of cervical fluid (CF) and menstrual cup collection of cervicovaginal fluid (CVF). Samples were collected at 3 time points across the second and third trimesters: 14-21, 22-25 and 26-31 weeks. Multiplex chemi-luminescent assays were used to measure: IFN-gamma, IL-1 beta, IL-2, IL-4, IL-6, IL-8, IL 10, IL-12, IL-13 and TNF-alpha. Optimal methodology for cytokine normalisation (sample weight, volume and total protein) was explored. Results: All cytokines were measurable in both fluid types. IL-beta, IL-8 and IL-6 were detected at the highest concentrations (ranking order CF > CVF > plasma). CVF collection was simpler, provided the largest volume of sample (median 0.5 g) with the potential for undiluted usage, and allowed for self-insertion. CF cytokine concentrations were intrinsically associated with sample weight and protein concentration however CVF cytokines were independent of these. Conclusion: Both methods of collection are robust for measurement of FGF cytokines during pregnancy. We recommend CVF collection using a menstrual cup as a viable option in pregnant women for high dimensional biological techniques.